Kenichiro Maeda

E-selectin engages PSGL-1 and CD44 through a common signaling pathway to induce integrin αLβ2-mediated slow leukocyte rolling

In inflamed venules, neutrophils rolling on E-selectin induce integrin alpha(L)beta(2)-dependent slow rolling on intercellular adhesion molecule-1 by activating Src family kinases (SFKs), DAP12 and Fc receptor-gamma (FcRgamma), spleen tyrosine kinase (Syk), and p38. E-selectin signaling cooperates with chemokine signaling to recruit neutrophils into tissues. Previous studies identified P-selectin glycoprotein ligand-1 (PSGL-1) as the essential E-selectin ligand and Fgr as the only SFK that initiate signaling to slow rolling. In contrast, we found that E-selectin engagement of PSGL-1 or CD44 triggered slow rolling through a common, lipid raft-dependent pathway that used the SFKs Hck and Lyn as well as Fgr. We identified the Tec kinase Bruton tyrosine kinase as a key signaling intermediate between Syk and p38. E-selectin engagement of PSGL-1 was dependent on its cytoplasmic domain to activate SFKs and slow rolling. Although recruiting phosphoinositide-3-kinase to the PSGL-1 cytoplasmic domain was reported to activate integrins, E-selectin-mediated slow rolling did not require phosphoinositide-3-kinase. Studies in mice confirmed the physiologic significance of these events for neutrophil slow rolling and recruitment during inflammation. Thus, E-selectin triggers common signals through distinct neutrophil glycoproteins to induce alpha(L)beta(2)-dependent slow rolling.

Interleukin-6 aborts lymphopoiesis and elevates production of myeloid cells in systemic lupus erythematosus–prone B6.Sle1.Yaa animals

We previously reported the inhibitory action of interleukin-6 (IL-6) on B lymphopoiesis with SHIP(-/-) mice and showed that IL-6 biases lineage commitment toward myeloid cell fates in vitro and in vivo. Because elevated IL-6 is a feature of chronic inflammatory diseases, we applied an animal model of systemic lupus erythematosus (SLE) to determine whether IL-6 has similar effects on hematopoiesis. We found that IL-6 levels were elevated in the B6.Sle1.Yaa mice, and the increase was accompanied by losses of CD19(+) B cells and more primitive B-lymphoid progenitors in bone marrow. Both the CD19(+) B-cell population and their progenitors recovered in an IL-6(-/-) background. The uncommitted progenitors, containing precursors for both lymphoid and myeloid fates, expressed IL-6 receptor-alpha chain and responded to IL-6 by phosphorylation of STAT3. IL-6 stimulation caused uncommitted progenitors to express the Id1 transcription factor, which is known to inhibit lymphopoiesis and elevate myelopoiesis, and its expression was MAPK dependent. We conclude that chronic inflammatory conditions accompanied by increased IL-6 production bias uncommitted progenitors to a myeloid fate by inducing Id1 expression.

IL-6 increases B-cell IgG production in a feed-forward proinflammatory mechanism to skew hematopoiesis and elevate myeloid production

Src homology 2 domain-containing inositol 5-phosphatase (SHIP(-/-)) animals display an age-related increase in interleukin-6 (IL-6), a decrease in B lymphopoiesis, and an elevation in myelopoiesis. We investigated the origin of the IL-6 production and show that it is largely produced by peritoneal and splenic macrophages. IL-6 production by these macrophages is not a direct result of the loss of SHIP: IL-6 production is not spontaneous, is absent from bone marrow-derived macrophages, declines with prolonged culture of macrophages, and requires a stimulus present in vivo. The IL-6-rich peritoneal cavity of SHIP(-/-) mice shows more than 700-fold more immunoglobulin G (IgG) than wild-type, approximately 20% of which is aggregated or in an immune complex and contains B220(+) cells that secrete IgG. The SHIP-deficient peritoneal macrophages show evidence of IgG receptor stimulation. Animals lacking both the signal-transducing gamma-chain of IgG receptors and SHIP or Ig and SHIP produce less IL-6. The data indicate a feed-forward process in which peripheral macrophages, responding through IgG receptors to secreted IgG, produce IL-6, to support further B-cell production of IgG. Because of the proinflammatory phenotype of SHIP(-/-) animals, these findings emphasize the importance of IL-6-neutralizing strategies in autoimmune and proinflammatory diseases.

Bacillus anthracis Peptidoglycan Stimulates an Inflammatory Response in Monocytes through the p38 Mitogen-Activated Protein Kinase Pathway

We hypothesized that the peptidoglycan component of B. anthracis may play a critical role in morbidity and mortality associated with inhalation anthrax. To explore this issue, we purified the peptidoglycan component of the bacterial cell wall and studied the response of human peripheral blood cells. The purified B. anthracis peptidoglycan was free of non-covalently bound protein but contained a complex set of amino acids probably arising from the stem peptide. The peptidoglycan contained a polysaccharide that was removed by mild acid treatment, and the biological activity remained with the peptidoglycan and not the polysaccharide. The biological activity of the peptidoglycan was sensitive to lysozyme but not other hydrolytic enzymes, showing that the activity resides in the peptidoglycan component and not bacterial DNA, RNA or protein. B. anthracis peptidoglycan stimulated monocytes to produce primarily TNFα; neutrophils and lymphocytes did not respond. Peptidoglycan stimulated monocyte p38 mitogen-activated protein kinase and p38 activity was required for TNFα production by the cells. We conclude that peptidoglycan in B. anthracis is biologically active, that it stimulates a proinflammatory response in monocytes, and uses the p38 kinase signal transduction pathway to do so. Given the high bacterial burden in pulmonary anthrax, these findings suggest that the inflammatory events associated with peptidoglycan may play an important role in anthrax pathogenesis.

Biological effect of dose distortion by fiducial markers in spot-scanning proton therapy with a limited number of fields: A simulation study

PURPOSE In accurate proton spot-scanning therapy, continuous target tracking by fluoroscopic x ray during irradiation is beneficial not only for respiratory moving tumors of lung and liver but also for relatively stationary tumors of prostate. Implanted gold markers have been used with great effect for positioning the target volume by a fluoroscopy, especially for the cases of liver and prostate with the targets surrounded by water-equivalent tissues. However, recent studies have revealed that gold markers can cause a significant underdose in proton therapy. This paper focuses on prostate cancer and explores the possibility that multiple-field irradiation improves the underdose effect by markers on tumor-control probability (TCP). METHODS A Monte Carlo simulation was performed to evaluate the dose distortion effect. A spherical gold marker was placed at several characteristic points in a water phantom. The markers were with two different diameters of 2 and 1.5 mm, both visible on fluoroscopy. Three beam arrangements of single-field uniform dose (SFUD) were examined: one lateral field, two opposite lateral fields, and three fields (two opposite lateral fields + anterior field). The relative biological effectiveness (RBE) was set to 1.1 and a dose of 74 Gy (RBE) was delivered to the target of a typical prostate size in 37 fractions. The ratios of TCP to that without the marker (TCP(r)) were compared with the parameters of the marker sizes, number of fields, and marker positions. To take into account the dependence of biological parameters in TCP model, α∕β values of 1.5, 3, and 10 Gy (RBE) were considered. RESULTS It was found that the marker of 1.5 mm diameter does not affect the TCPs with all α∕β values when two or more fields are used. On the other hand, if the marker diameter is 2 mm, more than two irradiation fields are required to suppress the decrease in TCP from TCP(r) by less than 3%. This is especially true when multiple (two or three) markers are used for alignment of a patient. CONCLUSIONS It is recommended that 1.5-mm markers be used to avoid the reduction of TCP as well as to spare the surrounding critical organs, as long as the markers are visible on x-ray fluoroscopy. When 2-mm markers are implanted, more than two fields should be used and the markers should not be placed close to the distal edge of any of the beams.

Evaluation of the relative biological effectiveness of spot-scanning proton irradiation in vitro

Variations in relative biological effectiveness (RBE) from a fixed value of 1.1 are critical in proton beam therapy. To date, studies estimating RBE at multiple positions relative to the spread-out Bragg peak (SOBP) have been predominantly performed using passive scattering methods, and limited data are available for spot-scanning beams. Thus, to investigate the RBE of spot-scanning beams, Chinese hamster fibroblast V79 cells were irradiated using the beam line at the Hokkaido University Hospital Proton Therapy Center. Cells were placed at six different depths, including the entrance of the proton beam and the proximal and distal part of the SOBP. Surviving cell fractions were analyzed using colony formation assay, and cell survival curves were obtained by the curve fitted using a linear–quadratic model. RBE10 and RBE37 were 1.15 and 1.21 at the center of the SOBP, respectively. In contrast, the distal region showed higher RBE values (1.50 for RBE10 and 1.85 for RBE37). These results are in line with those of previous studies conducted using passive scattering proton beams. Taken together, these data strongly suggest that variations in RBE should be considered during treatment planning for spot-scanning beams as well as for passive scattering proton beams.

A Nucleoside Anticancer Drug, 1-(3-C-Ethynyl-β-D-Ribo-Pentofuranosyl)Cytosine, Induces Depth-Dependent Enhancement of Tumor Cell Death in Spread-Out Bragg Peak (SOBP) of Proton Beam.

The effect of 1-(3-C-ethynyl-β-D-ribo-pentofuranosyl)cytosine (ECyd) on proton-induced cell death was evaluated in human lung carcinoma cell line A549 and Chinese hamster fibroblast cell line V79 to enhance relative biological effectiveness (RBE) within the spread-out Bragg peak (SOBP) of proton beams. Treatment with ECyd significantly enhanced the proton-induced loss of clonogenicity and increased senescence at the center, but not at the distal edge of SOBP. The p53-binding protein 1 foci formation assay showed that ECyd decelerated the rate of DNA double-strand break (DSB) repair at the center, but not the distal region of SOBP, suggesting that the ECyd-induced enhancement of proton-induced cell death is partially associated with the inhibition of DSB repair. This study demonstrated that ECyd enhances proton-induced cell killing at all positions of SOBP, except for the distal region and minimizes the site-dependent differences in RBE within SOBP. Thus, ECyd is a unique radiosensitizer for proton therapy that may be useful because it levels the biological dose within SOBP, which improves tumor control and reduces the risk of adverse effects at the distal edge of SOBP.

SU-F-T-131: No Increase in Biological Effectiveness Through Collimator Scattered Low Energy Protons

PURPOSE To reduce the lateral penumbra of low-energy proton beams, brass collimators are often used in spot-scanning proton therapy (SSPT). This study investigates the increase in biological effectiveness through collimator scattered protons in SSPT. METHODS The SSPT system of the Hokkaido University Hospital Proton Beam Therapy Center, which consists of a scanning nozzle, a 2-cm thick brass collimator, and a 4-cm thick energy absorber, was simulated with our validated Geant4 Monte Carlo code (ver. 9.3). A water phantom was irradiated with proton pencil beams of 76, 110, and 143 MeV. The tested collimator opening areas (COA) were 5×5, 10×10, and 15×15 cm2 . Comparisons were made among the dose-averaged LET values of protons that hit the collimators (LETDColl), protons that did not hit the collimators (LETDNoColl), and all protons (LETDTotal). X-ray equivalent doses (Deq) were calculated using the linear-quadratic model with LETDNoColl and LETDTotal, and their maximum difference was determined over regions where the physical dose was greater than 10% of the peak dose of 2 Gy. RESULTS The ratio of the dose contribution of collimator scattered protons to that of all protons, defined as λ, was large at high proton energies and large COAs. The maximum λ value ranged from 3% (76 MeV, 5×5 cm2 ) to 29% (143 MeV, 15×15 cm2 ). Moreover, a large difference between LETDColl and LETDNoColl was only found in regions where λ was below 20% (ΔLETD > 2 keV/µm) and 8% (ΔLETD > 5 keV/µm). Consequently, the maximum difference between LETDNoColl and LETDTotal was as small as 0.8 keV/µm in all simulated voxels, and the difference of Deq reached a maximum of 1.5% that of the peak dose obtained at the water surface with a 76 MeV beam. CONCLUSION Although collimator scattered protons have high LET, they only increase the physical dose, not the biological effectiveness.

MO‐F‐213AB‐04: Biological Effect of Dose Shadowing by Fiducial Markers in Spot Scanning Proton Therapy with a Limited Number of Fields

PURPOSE In spot scanning proton therapy, accurate patient positioning before and during treatment is essential. A small gold ball marker is suitable as a fiducial for prostate treatment. However, it has been pointed out that the marker causes dose shadowing because the protons are scattered with their energy quickly diminished. In this research we explore the possibility that the biological effect of dose shadowing can be mitigated with a limited number of fields. METHODS The proton dose distribution in prostate was simulated using Geant4. The simulations include the Hokkaido University spot scanning nozzle and a water phantom positioned isocentrically. The PTV was delineated at the center of the phantom and a gold ball of 2 mm in diameter was placed at the middle of the PTV. The plan was created by single-field optimization and each of the following beam arrangements was investigated; (1) single lateral field (2) two lateral fields (3) two lateral + one anterior fields (4) four-field box. The dose prescription was D95 = 74 GyE (37 fr). The minimum dose and tumor control probability (TCP) were compared for the four beam arrangements. RESULTS For (1)-(4), the minimum dose values were 55%, 77%, 78%, and 84% of the prescribed dose, respectively. The reduction of the TCP values from those in the absence of the gold marker were 50%, 2%, 1.1%, and 0.7%, using the TCP model by Wang et al. (Int.J.Radiat.Oncol.Biol.Phys. 55, 2003) and 2%, 0.7%, 0.5%, and 0.4%, using the biological parameters in Levegrün et al. (Int.J.RadiatOncol.Biol.Phys. 51, 2001), respectively. CONCLUSIONS Although dose shadowing by the gold marker is locally non-negligible, the size of the affected domain is tiny. It was found that with a minimum number of fields, the TCP nearly recovers to the value without the gold marker.